Neural circuit development requires neuronal processes to find the correct synaptic partners within a complex environment. Dynamic interactions between processes from different cell types often result in a reproducible layered organization that simplifies this connectivity problem. For example, in the Drosophila visual system the ultraviolet-sensitive R7 photoreceptor axons always connect to their post-synaptic Dm8 partners in the M6 layer of the medulla. Previous studies have identified factors required in R7 for its normal projection pattern, yet little is known about the cellular and molecular cues in the environment that it recognizes. The goal of this proposal is to close this gap by identifying the neurons and signals that shape the environment of the medulla and guide R7 through it. The first aim is based on the discovery that the CUB-LDL protein Lost and found (Loaf) is required in R7 only when it is also present in other cells in the environment. Determining the consequences of varying the levels of Loaf in different sets of neurons will distinguish between two hypotheses: that matching levels of Loaf in R7 and Dm8 promote synapse formation between them, or that R7 competes with other Loaf-expressing neurons for access to its target layer. Other experiments will investigate whether Loaf mediates homophilic or heterophilic adhesion or traffics other molecules to the synapse. The second aim will examine how Plexin A (PlexA) expressed on tangentially projecting neurons contributes to the separation of medulla layers. The proposed experiments will determine whether PlexA acts as a receptor to autonomously guide tangential neurons, which then provide guidance information to other processes, or as a ligand that is itself recognized by the ingrowing processes of medulla neurons and/or R7. The third aim will make use of a recent transcriptomic analysis to determine which of the cell-surface or secreted molecules that are enriched in Dm8 are necessary to promote synapse formation with R7. The most interesting candidate will be selected for further analysis of its mechanism of action. The cellular and molecular interactions defined in this study are likely to reveal principles that will be applicable to the assembly of more complex neural circuits, and to provide insight into the nature of the defects in neurodevelopmental disorders such as autism and epilepsy.